With emphasis on a single-cell approach, we are proposing experiments on antigen-specific T lymphocytes using a combination of optical techniques - confocal microscopy, two-photon microscopy, and optical tweezers - to investigate the dynamics of membrane receptor interactions during antigen presentation. Our laboratory is using biophysical techniques to investigate signaling mechanisms, biochemical and optical assays to monitor cell activation, and genetically modified cells to probe molecular mechanisms. The proposed experiments on T lymphocytes and antigen-presenting cells will focus on the dynamics of signaling from the engagement of membrane receptors to the [Ca2+]i signal. One goal of this project is to apply optical techniques to investigate antigen recognition, [Ca2+]i signaling, motility, and gene expression in T cells stimulated by physiological ligands. We will use reporter genes to visualize T-cell activation dynamically in order to relate membrane and se cond-messenger mechanisms to effector function. We are investigating interactions between cells during antigen presentation, as well as intracellular signaling mechanisms that link membrane receptors with gene expression.